Process for inhibiting activity of endotoxin

ABSTRACT

By contacting a peptide derivative or a protein having a property of binding to endotoxin (ET) to inhibit the activity of ET, and at least one surfactant with a sample containing ET, the activity of ET can be inhibited, and even when an ET analogous substance having ET activity is present in the thus treated sample, it can be measured without influence of ET.

This is a Divisional application of Ser. No. 08/183,436 filed Jan. 19,1994, now abandoned.

BACKGROUND OF THE INVENTION

This invention relates to a process for selective inhibition ofproperties of endotoxin (hereinafter abbreviated as "ET"), such as aproperty of reacting with horseshoe crab hemocyte lysate (hereinafterabbreviated as "AL solution") to cause activation reaction of an enzyme(e.g. protease) contained in said solution (hereinafter abbreviated as"enzyme activation reaction") or gelation reaction; and a process formeasuring at least one substance other than ET among substances havingET activity (hereinafter abbreviated as "activating substances for ALsolution") (the substance other than ET is hereinafter abbreviated as"ET analogous substance") which is contained in a sample treated by theabove inhibiting process.

ET's are lipopolysaccharides (LPS) present in cell walls ofGram-negative bacteria and are known as potent pyrogens. Therefore, thedetection of ET in parenteral drugs and the like is consideredimportant, and test methods for endotoxin are described in the U.S.Pharmacopoeia and the Japanese Pharmacopoeia. ET is considered a maincause of shock in Gram-negative bacterial infections. In clinicaldiagnoses, the measurement of endotoxin in plasma is employed, forexample, for diagnoses of Gram-negative bacterial infections, judgementof curative effect on and prognosis of Gram-negative bacterialinfections, and early diagnosis of endotoxin shock. AL solution has aproperty of being activated by ET to cause activation reaction of anenzyme (e.g. protease) or gelation reaction. Simple, low-cost ETdetecting methods utilizing this property, for example, so-calledLimulus test including a method of measuring the degree of activation ofthe enzyme (e.g. protease) by a colorimetric method, or utilizing thegelation reaction (in the present invention, the "Limulus test" includesthese methods mentioned above) are widely employed in the fields ofmedical science, pharmacy and microbiology.

Reagents used for the Limulus test, however, have been disadvantageousin that since they react also with an ET analogous substance(s) such as(1→3)-β-D-glucan and/or a derivative thereof (hereinafter abbreviated as"βG") [Kakinuma et al., Biochem. Biophys. Res. Commun., vol. 101,434-439 (1981), and Morita et al., FEBS Lett., vol. 129, 318-321(1981)], the presence of βG together with ET in a sample for measurementcauses positive errors in measured values.

Although, βG's are interfering substances which cause positive errors inET measurement, the detection of βG by use of AL solution is consideredutilizable for diagnoses of infectious diseases caused by Eumycetes,because they are components of cell wall of Eumycetes such as yeast andmold. Hence the development of a method for measurement of βG isinvestigated. However, since commercially available reagents usable forthe Limulus test, of course, react with ET, there has been a problem inthat when βG is measured using the reagents, ET in a sample affectsmeasured values.

For solving the above problems, there have been reported, for example, aprocess for removing a factor which is present in AL solution and reactswith ET to cause activation reaction of an enzyme (e.g. protease) orgelation reaction (hereinafter abbreviated as "ET-sensitive factor"), bytreating AL solution by various chromatographies (Japanese PatentUnexamined Publication (JP-A) No. 59-27828, JP-A H2-138193 and JP-AH4-76459), a process for inhibiting ET-sensitive factors by adding apeptide having affinity for ET to AL solution (JP-A H2-207098), and aprocess for inhibiting ET-sensitive factors by adding antibodies againstET-sensitive factor to AL solution (JP-A H4-52558). All of theseprocesses, however, are disadvantageous in that since they comprisetreating AL solution itself, there is a strong fear that during thetreatment, AL solution may be contaminated with ET or βG, which arewidely present in a usual environment. Moreover, these processes requiresterile facilities and complicated sterile operations for avoiding thecontamination, and the peptide having affinity for ET and the antibodiesagainst ET-sensitive factor are difficult to obtain and are expensive.As is clear from these facts and the like, the processes involve manyeconomical and technical problems.

For inactivating ET by treatment of not AL solution but a sample formeasurement, a process for heat-treating the sample has been reported(JP-A H2-141666). This process is free from the defects of the processesdescribed above, but is disadvantageous in that a long treatment time isrequired for sufficient inactivation of ET. Therefore, it cannot be saidto be a preferable process.

SUMMARY OF THE INVENTION

This invention was made in consideration of such conditions and isintended to provide a process for selective inhibition of ET activitywhich can be practiced by easy operations by use of easily availablereagents; a process for measuring at least one ET analogous substance ina sample by employing this inhibiting process; and a reagent for thismeasurement.

This invention provides a process for inhibiting the activity of ETwhich comprises contacting a peptide derivative or a protein, which hasa property of binding to ET to inhibit the activity of ET (hereinafterabbreviated as "ET-inhibiting peptide") and at least one surfactant witha sample containing ET.

This invention also provides a process for measuring at least one ETanalogous substance present in a sample which comprises reacting thesample treated by the above-mentioned inhibiting process with ALsolution, and measuring the activity of an enzyme activated by enzymeactivation reaction caused by the above reaction, or measuring thedegree of turbidity change or gelation of the reaction solution which isdue to gelation reaction caused by the above reaction, by means of ameasuring instrument or with the naked eye.

In addition, this invention provides a pretreating solution forinhibiting the activity of ET which is an aqueous solution containing anET-inhibiting peptide and at least one surfactant, does not react withAL solution, and neither inhibits nor enhance the reaction of ALsolution with an ET analogous substance.

Further, this invention provides a reagent for measuring at least one ETanalogous substance which comprises an ET-inhibiting peptide, at leastone surfactant and AL solution.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing the influence of the concentration of asurfactant on the measurement of endotoxin (hereinafter abbreviated as"ET") (E. coli 055: B5 LPS) in plasma, which was obtained in Example 1.

FIG. 2 is a graph showing the influence of the concentration ofpolymyxin B (hereinafter abbreviated as "Px B") on the measurement of ET(E. coli 055: B5 LPS) in plasma, which was obtained in Example 1.

FIG. 3 is a graph showing the influence of the concentration of thesurfactant on the measurement of ET (E. coli 0128: B12 LPS) in plasma,which was obtained in Example 4.

FIG. 4 is a graph showing the influence of the concentration of Px B onthe measurement of ET (E. coli 0128: B12 LPS) in plasma, which wasobtained in Example 4.

FIG. 5 is a graph showing the influence of the concentration of Px B onthe measurement of ET (E. coli 0128: B12 LPS) in plasma in the presenceof the surfactant, which was obtained in Example 4.

FIG. 6 is a graph showing the influence of the concentration of thesurfactant on the measurement of β-glucan in the presence of Px B, whichwas obtained in Example 9.

FIG. 7 is a graph showing the influence of the concentration of Px B onthe measurement of the ET concentration in an aqueous solutioncontaining ET (E. coli 055: B5 LPS), which was obtained in Example 10.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

In the course of earnest study for finding a process for specificmeasurement of an AL solution activating substance such as ET, βG, etc.by use of AL solution, the present inventors found that the presence ofboth a surfactant and an ET-inhibiting peptide in a sample or a reactionsolution obtained at the time of measuring the AL solution activatingsubstance inhibits only the activity of ET contained in the sample orthe reaction solution and permits specific detection of an ET analogoussubstance such as βG, whereby this invention has been accomplished.

In detail, it has been well known that surfactants and ET-inhibitingpeptides represented by polymyxin and the like have a property ofinhibiting ET activity. But, for inhibiting ET activity completely byusing a surfactant or an ET-inhibiting peptide alone, the addition of alarge amount of the surfactant or the ET-inhibiting peptide has beennecessary. Therefore, there has been a problem in that when an ETanalogous substance in a sample treated using either the surfactant orthe ET-inhibiting peptide alone is measured by the Limulus' test,influences such as inhibition of activation of enzymes in AL solutionare brought about, so that no exact measured value can be obtained.However, the present inventors found that simultaneous use of asurfactant and an ET-inhibiting peptide permits complete inhibition ofET activity in a sample even when they are added in such an amount thatthey do not affect the Limulus test, whereby this invention has beenaccomplished.

The surfactant used in this invention is not critical so long as itneither inhibits nor enhances the activation reactions (enzymeactivation reaction, gelation reaction, etc.) of AL solution by ETanalogous substances and it does not cause appearance of non-specificturbidity during the reactions.

Specific examples of the surfactant are nonionic surfactants such aspolyoxyethylene alkyl ethers (e.g. polyoxyethylene cetyl ether,polyoxyethylene lauryl ether, etc.), polyoxyethylene alkylphenyl ethers(e.g. polyoxyethylene octylphenyl ether, polyoxyethylene nonylphenylether, etc.), polyoxyethylene alkyl esters (e.g. polyoxyethylenesorbitan monooleate, polyoxyethylene sorbitan monopalmitate,polyoxyethylene sorbitan monostearate, polyoxyethylene sorbitantriolate, etc.), methylglucamide derivatives (e.g.octanoyl-N-methylglucamide, nonanoyl-N-methylglucamide,decanoyl-N-methylglucamide, etc.), alkyl sugar derivatives (e.g.n-octyl-β-D-glucoside, etc.) and the like; anionic surfactants such assodium dodecyl sulfate (SDS), laurylbenzenesulfonic acid, deoxycholicacid, cholic acid, tris(hydroxymethyl)aminomethane dodecyl sulfate (TrisDS) and the like; cationic surfactants such as alkylamine salts (e.g.octadecylamine acetic acid salt, tetradecylamine acetic acid salt,stearylamine acetic acid salt, laurylamine acetic acid salt,lauryldiethanolamine acetic acid salt, etc.), quaternary ammonium salts(e.g. octadecyltrimethylammonium chloride, dodecyltrimethylammoniumchloride, cetyltrimethylammonium chloride, cetyltrimethylammoniumbromide, allyltrimethylammonium methylsulfate, benzalkonium chloride,tetradecyldimethylbenzylammonium chloride,octadecyldimethylbenzylammonium chloride, lauryldimethylbenzylammoniumchloride, etc.), alkylpyridinium salts (laurylpyridinium chloride,stearylamidomethylpyridinium chloride, etc.) and the like; amphotericsurfactants such as3-[(3-cholamidoamidopropyl)dimethylammonio]-1-propane sulfonate,3-[(3-cholamidoamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonate and the like; and natural surfactants such as saponin (derivedfrom soybean), digitonin and the like. In view of influences on enzymesystems and the like during the reaction, the nonionic surfactants orthe amphoteric surfactants are preferable. The above-exemplifiedsurfactants may be used singly or in proper combination of two or morethereof.

Although their using concentration is varied depending on their kind anda sample to be treated, it may be chosen without particular limitationso long as it is a concentration at which ET activity can besufficiently inhibited in the presence of an ET-inhibiting peptide andthe reactivity of ET analogous substances such as βG with AL solution isneither enhanced nor inhibited. More specifically, the concentration ofthe surfactants in an ET-containing sample or a reaction solution ofsaid sample with AL solution is properly chosen usually in the range of0.005 to 5 w/v %, preferably 0.05 to 2.5 w/v %, more preferably 0.1 to 1w/v %.

The ET-inhibiting peptide used in this invention is not critical so longas it is a peptide derivative (or a protein) which has a property ofbinding to ET to inhibit the activity of ET. The ET-inhibiting peptidepreferably has properties of having strong basicity and/or havinghydrophobic moieties. Preferable examples of the ET-inhibiting peptideare polymyxin produced by Bacillus polymyxa or the like, andtachyplesin, polyphemusin, anti-LPS factor and the like which arederived from AL solution. Since tachyplesin, polyphemusin, anti-LPSfactor and the like are usually difficult to obtain and expensive,employment of polymyxin is economically advantageous. The polymyxin isnot particularly limited and polymyxins A, B, C, D, E, K, M, P and thelike may be used as a single purified component, a mixture of two ormore of them, or a salt of any of them. Sulfate of polymyxin B ispreferable in view of commercial availability.

Although the using concentration of the ET-inhibiting peptide is varieddepending on the kind of the ET-inhibiting peptide and a sample to betreated, it may be chosen without particular limitation so long as it isa concentration at which ET activity can be sufficiently inhibited inthe presence of the surfactant(s) and the reactivity of ET analogoussubstances such as βG with AL solution is neither enhanced norinhibited. More specifically, the concentration of the ET-inhibitingpeptide in an ET-containing sample or a reaction solution of said samplewith AL solution is properly chosen usually in the range of 0.00001 to0.1 w/v %, preferably 0.0001 to 0.05 w/v %, more preferably 0.001 to0.01 w/v %.

The inhibiting process of this invention is practiced, for example, asfollows.

The word "contacting" means that the endotoxin-inhibiting peptide andsurfactant contact with an ET-containing sample by means of addition,dilution, stirring, mixing, vibration, etc. For example, it issufficient that the above-exemplified surfactant(s) and ET-inhibitingpeptide are present in an ET-containing sample or at the time of thereaction of said sample with AL solution, at concentrations in theabove-mentioned ranges. Needless to say, when an ET analogous substanceis measured in a sample treated by the inhibiting process of thisinvention, the concentrations of the surfactant(s) and the ET-inhibitingpeptide at the time of the reaction of said sample and AL solution maybe below the above-mentioned ranges.

When the inhibiting process of this invention is employed for inhibitingET activity in a sample which may be heated, the ET activity can be moreefficiently inhibited by employing heat treatment together with theinhibiting process. Particularly when an ET analogous substance in asample containing inhibiting factors or the like, such as plasma orserum is measured by the Limulus test, it is preferable to employ heattreatment together with the inhibiting process as a pretreatment forremoving the influences of the inhibiting factors or the like. Althoughthe heat treatment may be carried out before or after the addition ofboth the surfactant(s) and the ET-inhibiting peptide, it is preferablycarried out after the addition because a larger effect can be oftenobtained. Although not critical, the heating temperature in the heattreatment is usually 60°-220° C. preferably approximately 70°-100° C.Although not critical, the heating time is usually 3-60 minutes,preferably approximately 5-15 minutes. Any heating method may beemployed so long as it permits heating of the sample under the aboveheating conditions. Preferable examples of heating method are heating inan incubator and heating in an autoclave.

For placing both the surfactant(s) and the ET-inhibiting peptide in anET-containing sample or at the time of the reaction of said sample withAL solution, any method may be employed so long as it is such that thesurfactant(s) and the ET-inhibiting peptide are added to said sample orAL solution to adjust their concentrations to values in theabove-mentioned ranges before initiation of the activation of ALsolution by ET. The following methods can be exemplified.

1) A method in which an ET-containing sample is properly diluted with asolution containing proper amounts of the surfactant(s) and theET-inhibiting peptide (or a solution containing a proper amount of thesurfactant(s) and a solution containing a proper amount of theET-inhibiting peptide) which does not activate AL solution and neitherinhibits nor enhance the reaction of AL solution with an ET analogoussubstance.

2) A method in which when an ET analogous substance is measured, properamounts of the surfactant(s) and the ET-inhibiting peptide arepreviously incorporated into AL solution, and the thus treated ALsolution is properly mixed with a sample for measurement.

When the above method 1) is employed, the degree of dilution of thesample is not critical, but when the sample is plasma or the like, thefollowing problems are caused in some cases. When the degree of dilutionis too low, an increase in viscosity or the formation of a precipitateis caused in the diluted plasma by denaturation of plasma protein duringheating. When the degree of dilution is too high, proper detection of ETbecomes impossible. Therefore, when the sample is plasma or the like,the degree of dilution is usually 5 to 20 times, preferably about 8 toabout 12 times.

As the solution or solutions used in the above method 1), i.e., thesolution containing the surfactant(s) and the ET-inhibiting peptide orthe solutions containing them respectively, an aqueous solution of theabove-exemplified surfactant(s) and ET-inhibiting peptide or aqueoussolutions of them respectively are prepared so as to have a properconcentrations(s), and are used preferably after confirming that theaqueous solution(s) does not activate AL solution and neither enhancesnor inhibits the reaction in the Limulus test. The solution containingthe surfactant(s) and the ET-inhibiting peptide or the solutionscontaining them respectively may be autoclaved at 121° C. for 20 minutesfor sterilization. Although the concentration of the surfactant(s) inthe solution is not critical so long as it is in a range in which thefinal concentration of the surfactant(s) in a dilution obtained bydiluting an objective sample with the solution(s) is in the rangedescribed above, it is usually 0.01 to 10 w/v %, preferably 0.1 to 5 w/v%, more preferably 0.2 to 2 w/v %, in view of ease of operations.Although the concentration of the ET-inhibiting peptide in the solutionis not critical so long as it is in a range in which the finalconcentration of the ET-inhibiting peptide in a dilution obtained bydiluting an objective sample with the solution(s) is in the rangedescribed above, it is usually 0.00002 to 0.2 w/v %, preferably 0.0002to 0.1 w/v %, more preferably 0.002 to 0.02 w/v %, in view of ease ofoperations.

The AL solution containing predetermined amounts of the surfactant(s)and the ET-inhibiting peptide which is used in the above method 2) can,of course, be once freeze-dried, stored in the form of a reagent,redissolved in water containing no AL solution activating substance,such as distilled water for injection, and then used.

Since the inhibiting process of this invention makes it possible toinhibit ET activity in a sample completely by simultaneous use of smallamounts of the surfactant(s) and the ET-inhibiting peptide, said processis very economical. A sample treated by the inhibiting process of thisinvention has low contents of the surfactant(s) and the ET-inhibitingpeptide and hence can be used as a sample for measuring an ET analogoussubstance by utilization of Limulus test, without considering theinfluences of the surfactant(s) and the ET-inhibiting peptide on Limulustest.

Depending on the kind (source) of ET, its ET activity can besufficiently inhibited using a low concentration of the ET-inhibitingpeptide alone. Such inhibition is possible, for example, in the case ofET derived from E. coli 0127: B8. However, when the inhibition of ETactivity is necessary for measuring an ET analogous substance in asample in which it is not clear what kind of ET is contained,application of the inhibiting process of this invention is, of course,preferable.

The process for inhibiting ET activity of the present invention can beapplied, for example, in the following fields.

In the case of measuring the amount of ET analogous substance such asβ-glucan (more precisely (1→3)-β-D-glucan) in a sample using the Limulustest, the ET activity inhibiting process can be used to inhibitinfluences of endotoxin present in the sample on the results ofmeasurement.

Further, in the case of measuring endotoxin or endotoxin analogoussubstance such as β-glucan in a sample by adding an AL solution to thesample to bring about the activation of the AL solution, followed byaddition of a synthetic substrate thereto, the ET activity inhibitingprocess can be used to remove contamination of used reagents such as asynthetic substrate with endotoxin.

In addition, in an experiment using cultured cells, etc., when thecultured cells are susceptible to influences of endotoxin, the ETactivity inhibiting process can be used to remove the contamination ofused reagents with endotoxin.

To measure an ET analogous substance in a sample treated by theinhibiting process of this invention, it is sufficient that themeasurement is carried out by a conventional method using AL solution,for example, the so-called Limulus test, i.e., a kinetic turbidimetrictechnique using an apparatus for exclusive use, such as ToxinometerET-201 (mfd. by Wako Pure Chemical Industries, Ltd.), Toxinometer MT-251(mfd. by Wako Pure Chemical Industries, Ltd.), LAL-5000 [mfd. byAssociates of Cape Cod Inc. (ACC)] or the like; a chromogenic techniquecomprising measuring protease activity which arises owing to theactivation of AL solution, by use of a synthetic substrate; or agel-clot technique comprising judging whether a gel is produced by theactivation of AL solution or not with the naked eye.

The ET analogous substance according to this invention is not criticalso long as it is other than ET and reacts with AL solution to causeenzyme activation reaction or gelation reaction. Typical examples of theET analogous substance are (1→3)-β-D-glucan and derivatives thereof. Asthe (1→3)-β-D-glucan and derivatives thereof, any polysaccharides can beexemplified without particular limitation so long as they contain(1→3)-β-D-glucoside linkage. Preferable examples of the (1→3)-β-D-glucanare natural polysaccharides obtained from cell walls or other componentsof, for example, various bacteria (e.g. Alcaligenes genus, Agrobacteriumgenus, etc.), yeasts (e.g. Saccharomyces genus, Candida genus,Crytrococcus genus, Trichosporon genus, Rhodotorula genus, etc.), molds(e.g. Aspergillus genus, Mucor genus, Penicillium genus, Trichophyrongenus, Sporothrix genus, Phialophora genus, etc.), actinomycetes(Actinomyces genus, Nocardia genus,etc.), and mushrooms (e.g.Cortinellus shiitake, Schizophyrum commune, Coriolus versicolor, etc.),specific examples of the natural polysaccharides being curdlan,pachyman, sclerotan, lentinan, schizophyllan, coriolan, etc.; storagepolysaccharides of algae, e.g. brown algae, Euglena, diatoms, etc.,specific examples of the storage polysaccharides being laminaran,paramylon, etc. Preferable examples of the (1→3)-β-D-glucan derivativesare polysaccharide derivatives obtained by introducing at least onegroup selected from the group consisting of a sulphonic group,carboxymethyl group, carboxyethyl group, methyl group, hydroxyethylgroup, hydroxy propyl group, sulfopropyl group, etc. into the naturalpolysaccharides or the storage polysaccharides according to aconventional method, for instance, any of the methods described, forexample, in Munio Kotake "Daiyukikagaku" vol. 19, 7th ed. AsakuraShoten, May 10, 1967, pp. 70-101; A. E. Clarke et al., Phytochemistry,vol. 1, 175-188 (1967); and T. Sasaki et al. Europ. J. Cancer, vol. 15,211-215 (1967).

The AL solution usable in this invention is not critical so long as itcan be used for usual measurement of ET. There may be used AL solutionsprepared from freeze-dried products of AL solutions which arecommercially available, for example, from Associates of Cape Cod Inc.(ACC), HAEMACHEM, Inc., Biowhittaker Inc., Endosafe Inc., Teikoku HormonMfg. Co., Ltd., and Seikagaku Kogyo Co., Ltd. In addition, there can beexemplified any AL solution without particular limitation so long as itis prepared from hemocytes of horseshoe crab belonging to Limulus genus,Tachypleus genus or Carcinoscorpius genus and react with an AL solutionactivating substance to undergo enzyme (protease or the like) activationor gelation reaction.

This invention is illustrated below in further detail with reference toExamples, which are not by way of limitation but by way of illustration.

EXAMPLE 1

[Reagents]

ET solutions

There were used solutions prepared by weighing 10 mg of Escherichia coli(E. coli) 055: B5 LPS (available from Difco Laboratories), dissolvingthe same in 10 ml of distilled water for injection to prepare a 1 mg/mlsolution as a starting solution, and diluting the starting solutionproperly with distilled water for injection.

AL solution

A freeze-dried product of AL solution derived from horseshoe crabbelonging to Limulus genus (hereinafter the freeze-dried product beingabbreviated as "LAL"; available from Wako Pure Chemical Industries,Ltd.; for dissolution in 2 ml) was dissolved in a buffer solution forLAL reconstitution (HS) (mfd. by Wako Pure Chemical Industries, Ltd.),and the LAL solution thus obtained was used as AL solution.

Aqueous surfactant solutions

Polyoxyethylene glycol p-t-octylphenyl ether (a nonionic surfactant,mfd. by Wako Pure Chemical Industries, Ltd.) was diluted to 10 w/v %with distilled water for injection, and the resulting dilution wasautoclaved at 121° C. for 20 minutes to obtain a starting solution. Thestarting solution was properly diluted with distilled water forinjection, and the thus obtained diluted solutions were used as aqueoussurfactant solutions after confirming that they did not activate the ALsolution and neither inhibited nor enhance the reaction for measuring βGby the Limulus test.

Aqueous polymyxin B (hereinafter abbreviated as "Px B") solutions

Px B sulfate (mfd. by Wako Pure Chemical Industries, Ltd.) was dilutedto 1.0 w/v % with distilled water for injection, and the resultingdilution was autoclaved at 121° C. for 20 minutes to obtain a startingsolution. The starting solution was properly diluted with distilledwater for injection (or 0.2 w/v % aqueous surfactant solution preparedin the above), and the thus obtained dilutions were used as aqueous Px Bsolutions after confirming that they did not activate the AL solutionand neither inhibited nor enhance the reaction for measuring βG by theLimulus test.

[Procedure]

To 2.0 ml of normal human heparinized plasma was added 20 μl of ETsolution having a predetermined concentration, and 100 μl of theresulting ET spiked plasma was diluted 10 times (i.e. 1/10) with 900 μlof distilled water for injection, each of aqueous Px B solutions havingpredetermined concentrations, or each of aqueous surfactant solutionshaving predetermined concentrations. Each of the thus obtained dilutionswas heat-treated at 80° C. for 5 minutes and then immediately cooledwith ice (the final concentrations of ET, Px B and the surfactant were 2ng/ml, 0.0018 to 0.009 w/v % and 0.09 to 0.45 w/v %, respectively). TheET concentration in the diluted plasma samples thus obtained wasmeasured as follows by a conventional method using an apparatus"Toxinometer MT-251" (mfd. by Wako Pure Chemical Industries, Ltd.).

To 0.1 ml of the LAL solution was added 0.1 ml to each of the aforesaiddiluted plasma samples, and stirred, after which a time required forreducing the transmittance of the resulting mixture by 5% (hereinafterabbreviated as "Tg") was measured while maintaining the temperature at37° C. The ET concentration in the diluted plasma samples was determinedon the basis of the thus obtained Tg value by use of a calibration curveshowing the relationship between ET concentration and Tg which had beenpreviously obtained by carrying out the same measurement as describedabove except for using ET solutions varies in their concentrations assamples diluted plasma various in their concentrations as samplesdiluted plasma.

[Results]

FIGS. 1 and 2 show the results obtained. FIG. 1 shows the resultsobtained for the plasma diluted with each surfactant solution and is agraph obtained by plotting the gelation time (Tg) of the diluted andheated plasma on the axis of ordinate corresponding to individualsurfactant concentrations (at the time of the heat treatment) on theaxis of abscissa. FIG. 2 shows the results obtained for the plasmadiluted with each Px B solution and is a graph obtained by plotting thegelation time (Tg) of the diluted and heated plasma on the axis ofordinate corresponding to individual Px B concentrations (at the time ofthe heat treatment) on the axis of abscissa. In FIG. 2, -Δ- shows theresults obtained by use of the aqueous solutions containing Px B alone,and -□- shows the results obtained by use of the aqueous solutionscontaining both Px B and 0.2 w/v % of the surfactant (polyoxyethyleneglycol p-t-octylphenyl ether) (the surfactant concentration at the timeof the heat treatment was 0.18 w/v %). In FIG. 2, (□) indicates that Tgwas 90 minutes or more, in other words, ET was not detected.

As is clear from the results shown in FIG. 1, the Tg value obtained fromthe plasma diluted with distilled water for injection was 7.8 minutes,while the Tg values obtained from the plasma diluted with each of 0.1 to0.5% aqueous surfactant solutions (the surfactant concentration at thetime of the heat treatment was 0.09 to 0.45%) was as large as 16.3 to33.7 minutes, indicating that the ability of ET to activate AL solution(ET activity) was deteriorated (the smaller Tg, the higher the ETactivity). As is clear from the results shown in FIG. 2, the Tg valueobtained from the plasma diluted with distilled water for injection was7.8 minutes, while the Tg values obtained from the plasma diluted witheach of 0.002 to 0.01 w/v % aqueous Px B solutions (the Px Bconcentration at the time of the heat treatment was 0.0018 to 0.009 w/v%) was as large as 24.2 to 32.9 minutes, indicating that the ET activitywas lowered. Further, from the results shown in FIG. 2, it can be seenthat when plasma was diluted with aqueous solution containing both 0.002to 0.01 w/v % of Px B and 0.2% of the surfactant (the concentrations ofthe surfactant and Px B at the time of the heat treatment were 0.18% and0.0018 to 0.009 w/v %, respectively), ET was not detected (Tg became 90minutes or more).

As is clear from the results described above, the ET activity of E. coli0.55: B5 LPS spiked to plasma is completely inhibited by thesimultaneous presence of the surfactant and Px B.

EXAMPLE 2

[Reagents]

The same AL solution, aqueous surfactant solutions and aqueous Px Bsolutions as described in Example 1 were used.

ET solutions

There were used solutions prepared by weighing 10 mg of E. coli 0111: B4LPS (available from Difco Laboratories), dissolving the same in 10 ml ofdistilled water for injection to prepare a 1 mg/ml solution as astarting solution, and diluting the starting solution properly withdistilled water for injection.

[Procedure]

To 0.6 ml of normal human heparinized plasma was added 12 μl of ETsolution having a predetermined concentration, and 100 μl of theresulting ET spiked plasma was diluted 10 times with 900 μl of distilledwater for injection, 0.04 w/v % aqueous surfactant solution, 0.2 w/v %aqueous surfactant solution, or 0.01 w/v % aqueous Px B solution[containing 0.2 w/v % of the surfactant (polyoxyethylene glycolp-t-octylphenyl ether)], respectively. Each of the thus obtaineddilutions was heat-treated at 80° C. for 5 minutes and then immediatelycooled with ice (the final concentration of ET was 1.96 ng/ml). The ETconcentration in the diluted plasma thus obtained was measured in thesame manner as in Example 1.

[Results]

The results obtained are shown in Table 1.

                  TABLE 1                                                         ______________________________________                                                                   Concentration                                      Diluent           Tg (min) (pg/ml)                                            ______________________________________                                        Distilled water for                                                                             10.0     289.1                                              injection                                                                     0.04% Aqueous surfactant                                                                        6.3      2363.0                                             solution                                                                      0.2% Aqueous surfactant                                                                         40.4     3.4                                                solution                                                                      0.01% Aqueous Px B                                                                              >90      --                                                 solution (containing 0.2%                                                     of the surfactant)                                                            ______________________________________                                    

As is clear from the results shown in Table 1, the Tg value obtainedfrom the plasma diluted with distilled water for injection was 10.0minutes, while the Tg value obtained from the plasma diluted with the0.2 w/v % aqueous surfactant solution (the surfactant concentration atthe time of the heat treatment was 0.18 w/v %) was as large as 40.4minutes, indicating that the ET activity was lowered. It can also beseen that when plasma was diluted with the 0.01 w/v % aqueous Px Bsolution (containing 0.2 w/v % of the surfactant) (the concentrations ofPx B and the surfactant at the time of the heat treatment were 0.009 w/v% and 0.18 w/v %, respectively), ET was not detected (Tg became 90minutes or more). In the case of the plasma diluted with the 0.04%aqueous surfactant solution, the Tg value was smaller (the ET activitywas higher) than in the case of the plasma diluted with distilled waterfor injection. The reason can be presumed as follows: the influence offactors capable of inhibiting the reaction of ET with AL solution whichwere present in plasma was reduced by the addition of the surfactant.

As is clear from the results described above, the ET activity of E. coli0.111: B4 LPS spiked to plasma is completely inhibited by thesimultaneous presence of the surfactant and Px B.

EXAMPLE 3

[Reagents]

The same AL solution, aqueous surfactant solutions and aqueous Px Bsolutions as described in Example 1 were used.

ET solutions

There were used solutions prepared by weighing 10 mg of E. coli 0127: B8LPS (available from Difco Laboratories), dissolving the same in 10 ml ofdistilled water for injection to prepare a 1 mg/ml solution as astarting solution, and diluting the starting solution properly withdistilled water for injection.

[Procedure]

To 0.7 ml of normal human heparinized plasma was added 14 μl of ETsolution having a predetermined concentration, and 100 μl of theresulting ET spiked plasma was diluted 10 times with 900 μl of distilledwater for injection, 0.04 w/v % aqueous surfactant solution, 0.2 w/v %aqueous surfactant solution, 0.01 w/v % aqueous Px B solution, or 0.01w/v % aqueous Px B solution containing 0.2 w/v % of the surfactant(polyoxyethylene glycol p-t-octylphenyl ether), respectively. Each ofthe thus obtained diluted solutions was heat-treated at 80° C. for 5minutes and then immediately cooled with ice (the final concentration ofET was 1.96 ng/ml). The ET concentration in the diluted plasma thusobtained was measured in the same manner as in Example 1.

[Results]

The results obtained are shown in Table 2.

                  TABLE 2                                                         ______________________________________                                                                   Concentration                                      Diluent           Tg (min) (pg/ml)                                            ______________________________________                                        Distilled water for                                                                             10.6     221.2                                              injection                                                                     0.04% Aqueous surfactant                                                                        6.6      2152.0                                             solution                                                                      0.2% Aqueous surfactant                                                                         28.6     6.3                                                solution                                                                      0.01% Aqueous Px B                                                                              >90      --                                                 solution                                                                      0.01% Aqueous Px B                                                                              >90      --                                                 solution (containing 0.2%                                                     of the surfactant)                                                            ______________________________________                                    

As is clear from the results shown in Table 2, the Tg value obtainedfrom the plasma diluted with distilled water for injection was 10.6minutes, while the Tg value obtained from the plasma diluted with the0.2 w/v % aqueous surfactant solution (the surfactant concentration atthe time of the heat treatment was 0.18 w/v %) was as large as 28.6minutes, indicating that the ET activity was lowered. It can also beseen that when plasma was diluted with the 0.01 w/v % aqueous Px Bsolution or the 0.01 w/v % aqueous Px B solution containing 0.2 w/v % ofthe surfactant (the concentrations of Px B and the surfactant at thetime of the heat treatment were 0.009 w/v % and 0.18 w/v %,respectively), ET was not detected (Tg became 90 minutes or more). Inthe case of the plasma diluted with the 0.04% aqueous surfactantsolution, the Tg value was smaller (the ET activity was higher) than inthe case of the plasma diluted with distilled water for injection. Thereason can be presumed as follows: the influence of factors capable ofinhibiting the reaction of ET with AL solution which were present inplasma was reduced by the addition of the surfactant.

As is clear from the results described above, the ET activity of E. coli0.127: B8 LPS spiked to plasma is completely inhibited by thesimultaneous presence of the surfactant and Px B.

EXAMPLE 4

[Reagents]

The same AL solution, aqueous surfactant solutions and aqueous Px Bsolutions as described in Example 1 were used.

ET solutions

There were used solutions prepared by weighing 10 mg of E. coli 0128:B12 LPS (available from Difco Laboratories), dissolving the same in 10ml of distilled water for injection to prepare a 1 mg/ml solution as astarting solution, and diluting the starting solution properly withdistilled water for injection.

[Procedure]

To 1.3 ml of normal human heparinized plasma was added 13 μl of ETsolution having a predetermined concentration, and 100 μl of theresulting ET spiked plasma was diluted 10 times with 900 μl of distilledwater for injection, each of aqueous surfactant solutions havingpredetermined concentrations, each of aqueous Px B solutions havingpredetermined concentrations, or each of aqueous Px B solutions havingpredetermined concentrations and containing 0.1 w/v % or 0.2 w/v % ofthe surfactant (polyoxyethylene glycol p-t-octylphenyl ether),respectively. Each of the thus obtained dilutions was heat-treated at80° C. for 5 minutes and then immediately cooled with ice (the finalconcentrations of ET, Px B and the surfactant were 2 ng/ml, 0.00009 to0.009 w/v %, and 0.09 to 0.9 w/v %, respectively). The ET concentrationin the diluted plasma thus obtained was measured in the same manner asin Example 1.

[Results]

FIGS. 3 to 5 show the results obtained. FIG. 3 shows the resultsobtained from the plasma diluted with each surfactant solution and is agraph obtained by plotting the gelation time (Tg) of the diluted andheated plasma on the axis of ordinate corresponding to individualsurfactant concentrations (at the time of the heat treatment) on theaxis of abscissa. FIG. 4 shows the results obtained from the plasmadiluted with each Px B solution and is a graph obtained by plotting thegelation time (Tg) of the diluted and heated plasma on the axis ofordinate corresponding to individual Px B concentrations (at the time ofthe heat treatment) on the axis of abscissa. FIG. 5 shows the resultsobtained from the plasma diluted with each aqueous solution containingthe surfactant and Px B and is a graph obtained by plotting the gelationtime (Tg) of the diluted and heated plasma on the axis of ordinatecorresponding to individual Px B concentrations (at the time of the heattreatment) on the axis of abscissa. In FIG. 5, -□- shows the resultsobtained by use of aqueous solutions containing 0.1 w/v % of thesurfactant (the surfactant concentration at the time of the heattreatment was 0.09 w/v %), and -x- shows the results obtained by use ofaqueous solutions containing 0.2 w/v % of the surfactant (the surfactantconcentration at the time of the heat treatment was 0.18 w/v %). All of(∘) in FIG. 4 and (□) and (x) in FIG. 5 indicate that Tg was 90 minutesor more, in other words, ET was not detected.

As is clear from the results shown in FIGS. 3 to 5, the ET activity inplasma can be completely inhibited by the presence of 0.009 w/v % of PxB or the simultaneous presence of the surfactant and Px B. It can alsobe seen that when the ET activity is inhibited by the inhibiting processof this invention, the amount of Px B used can be reduced (to onehundredth that required in the case of using Px B alone), in otherwords, the process of this invention is economically superior toconventional processes using Px B alone.

EXAMPLE 5

[Reagents]

The same AL solution, aqueous surfactant solutions and aqueous Px Bsolutions as described in Example 1 were used.

ET solutions

There were used solutions prepared by weighing 10 mg of Salmonellatyphimurium LPS (available from Difco Laboratories), dissolving the samein 10 ml of distilled water for injection to prepare a 1 mg/ml solutionas a starting solution, and diluting the starting solution properly withdistilled water for injection.

[Procedure]

To 1.0 ml of normal human heparinized plasma was added 10 μl of ETsolution having a predetermined concentration, and 100 μl of theresulting ET spiked plasma was diluted 10 times with 900 μl of distilledwater for injection, 0.2 w/v % aqueous surfactant solution, 0.01 w/v %aqueous Px B solution, or 0.01 w/v % aqueous Px B solution containing0.2 w/v % of the surfactant (polyoxyethylene glycol p-t-octylphenylether), respectively. Each of the thus obtained dilutions washeat-treated at 80° C. for 5 minutes and then immediately cooled withice (the final concentration of ET was 2 ng/ml). The ET concentration inthe diluted plasma thus obtained was measured in the same manner as inExample 1.

[Results]

The results obtained are shown in Table 3.

                  TABLE 3                                                         ______________________________________                                                                   Concentration                                      Diluent           Tg (min) (pg/ml)                                            ______________________________________                                        Distilled water for                                                                             8.2      453.6                                              injection                                                                     0.2% Aqueous surfactant                                                                         12.3     77.9                                               solution                                                                      0.01% Aqueous Px B                                                                              13.4     55.7                                               solution                                                                      0.01% Aqueous Px B                                                                              >90      --                                                 solution (containing 0.2%                                                     of the surfactant)                                                            ______________________________________                                    

As is clear from the results shown in Table 3, the ET activity of S.typhimurium LPS spiked to plasma is completely inhibited by thesimultaneous presence of the surfactant and Px B.

EXAMPLE 6

[Reagents]

The same AL solution, aqueous surfactant solutions and aqueous Px Bsolutions as described in Example 1 were used.

Aqueous β-glucan solutions

There were used solutions prepared by weighing 100 mg ofcarboxymethylated curdlan [a linear (i+3)-β-D-glucan derivative, mfd. byWako Pure Chemical Industries, Ltd.], dissolving the same in 10 ml ofdistilled water for injection to prepare a 10 mg/ml solution as astarting solution, and diluting the starting solution properly withdistilled water for injection.

[Procedure]

By diluting 100 μl of 50 ng/ml aqueous βglucan solution 10 times with900 μl of 0.2 w/v % aqueous surfactant solution, 0.01 w/v % aqueous Px Bsolution, or 0.01 w/v % aqueous Px B solution containing 0.2 w/v % ofpolyoxyethylene glycol p-t-octylphenyl ether, solutions containingspiked β-glucan (the final concentration of β-glucan was 5 ng/ml) wereprepared. The β-glucan concentration in each of the prepared solutionswas measured as follows by a conventional method using an apparatus"Toxinometer MT-251" (mfd. by Wako Pure Chemical Industries, Ltd.). To0.1 ml of the LAL solution was added 0.1 ml of each of theabove-mentioned diluted solutions, and stirred, after which a timerequired for reducing the transmittance of the resulting mixture by 5%(hereinafter abbreviated as "Tg") was measured while maintaining thetemperature at 37° C. The β-glucan concentration was determined on thebasis of the thus obtained Tg value by use of a calibration curveshowing the relationship between β-glucan concentration and Tg which hadbeen previously obtained by carrying out the same measurement asdescribed above except for using β-glucan solutions various in theirconcentrations as samples. On the basis of the results of themeasurement, the recovery of β-glucan was calculated.

[Results]

The results obtained are shown in Table 4.

                  TABLE 4                                                         ______________________________________                                                            Recovery of                                               Diluent             β-glucan (%)                                         ______________________________________                                        0.2% Aqueous surfactant                                                                           100                                                       solution                                                                      0.01% Aqueous Px B solution                                                                       112                                                       0.01% Aqueous Px B solution                                                                        88                                                       (containing 0.2% of the                                                       surfactant)                                                                   ______________________________________                                    

As is clear from the results shown in Table 4, the recovery of β-glucanwas as high as about 90% to about 110%, whichever diluted solution wasused. Therefore, it can be seen that none of the diluted solutionseither inhibit or enhance the reaction of AL solution with β-glucan.

EXAMPLE 7

[Reagents]

The same AL solution, aqueous surfactant solutions and aqueous Px Bsolutions as described in Example 1 were used.

ET solutions

There were used solutions prepared by weighing 10 mg of E. coli 026: B6LPS (available from Difco Laboratories), dissolving the same in 10 ml ofdistilled water for injection to prepare a 1 mg/ml solution as astarting solution, and diluting the starting solution properly withdistilled water for injection.

Aqueous surfactant solutions

Various aqueous surfactant solutions were prepared in the same manner asdescribed in Example 1 except for using as surfactants, sodiumdeoxycholate (an anionic surfactant, mfd. by Wako Pure ChemicalIndustries, Ltd.), Emalgen 709 [a nonionic surfactant (a polyoxyethylenehigher alcohol ether), a trade name, Kao Corp.] and Amphitol 20N (anamphoteric surfactant, a trade name, Kao Corp.; main constituent:lauryldimethylamine oxide).

[Procedure]

To 1.7 ml of normal human heparinized plasma was added 17 μl of ETsolution having a predetermined concentration, and 100 μl of theresulting ET spiked plasma was diluted 10 times with 900 μl of distilledwater for injection, 0.1 w/v % aqueous sodium deoxycholate solution, 0.2w/v % aqueous Emalgen 709 solution, 0.4 w/v % aqueous Amphitol 20Nsolution, 0.01 w/v % aqueous Px B solution, 0.1 w/v % aqueous sodiumdeoxycholate solution containing 0.01 w/v % of Px B, 0.2 w/v % aqueousEmalgen 709 solution containing 0.01 w/v % of Px B, or 0.4 w/v % aqueousAmphitol 20N solution containing 0.01 w/v % of Px B. Each of the thusobtained diluted solutions was heat-treated at 80° C for 5 minutes andthen immediately cooled with ice (the final concentration of ET was 4ng/ml). The ET concentration in the diluted plasma thus obtained wasmeasured in the same manner as in Example 1.

[Results]

The results obtained are shown in Table 5.

                  TABLE 5                                                         ______________________________________                                                                   Concentration                                      Diluent          Tg (min)  (pg/ml)                                            ______________________________________                                        Distilled water for                                                                            9.0       1019.0                                             injection                                                                     0.1% Aqueous sodium                                                                            7.5       2287.0                                             deoxycholate solution                                                         0.2% Aqueous Emalgen 709                                                                       27.3      22.7                                               solution                                                                      0.4% Aqueous Amphitol 20N                                                                      52.6      4.3                                                solution                                                                      0.01% Aqueous Px B                                                                             30.8      16.2                                               solution                                                                      0.1% Aqueous sodium                                                                            >90       --                                                 deoxycholate solution                                                         (containing 0.01% of Px B)                                                    0.2% Aqueous Emalgen 709                                                                       >90       --                                                 solution (containing 0.01%                                                    of Px B)                                                                      0.4% Aqueous Amphitol 20N                                                                      >90       --                                                 solution (containing 0.01%                                                    of Px B)                                                                      ______________________________________                                    

As is clear from the results shown in Table 5, the ET activity of E.coli 0.26: B6 LPS spiked to plasma is completely inhibited by thesimultaneous presence of each surfactant and Px B.

EXAMPLE 8

[Reagents]

The same AL solution, aqueous surfactant solutions and aqueous Px Bsolutions as described in Example 1 were used.

Aqueous surfactant solutions

Various aqueous surfactant solutions were prepared in the same manner asdescribed in Example 1 except for using as surfactants, polyoxyethyleneglycol p-t-octylphenyl ether (a nonionic surfactant, mfd. by Wako PureChemical Industries, Ltd.), Emalgen 709 [a nonionic surfactant (apolyoxyethylene higher alcohol ether), a trade name, Kao Corp.] andAmphitol 20N (an amphoteric surfactant, a trade name, Kao Corp.; mainconstituent: lauryldimethylamine oxide).

ET solutions

There were used solutions prepared by weighing 10 mg of Salmonellatyphosa 0901 LPS (available from Difco Laboratories), dissolving thesame in 10 ml of distilled water for injection to prepare a 1 mg/mlsolution as a starting solution, and diluting the starting solutionproperly with distilled water for injection.

[Procedure]

To 1.0 ml of normal human heparinized plasma was added 10 μl of ETsolution having a predetermined concentration, and 100 μl of theresulting ET spiked plasma was diluted 10 times with 900 μl of distilledwater for injection, 0.2 w/v % aqueous polyoxyethylene glycolp-t-octylphenyl ether solution containing 0.01 w/v % of Px B, 0.2 w/v %aqueous Emalgen 709 solution containing 0.01 w/v % of Px B, or 0.4 w/v %aqueous Amphitol 20N solution containing 0.01 w/v % of Px B. Each of thethus obtained diluted solutions was heat-treated at 80° C. for 5 minutesand then immediately cooled with ice (the final concentration of ET was4 ng/ml). The ET concentration in the diluted plasma thus obtained wasmeasured in the same manner as in Example 1.

[Results]

The results obtained are shown in Table 6.

                  TABLE 6                                                         ______________________________________                                                                    Concentration                                     Diluent           Tg (min)  (pg/ml)                                           ______________________________________                                        Distilled water for                                                                             7.8       910.9                                             injection                                                                     0.2% Aqueous polyoxyethylene                                                                    >90       --                                                glycol p-t-octylphenyl ether                                                  solution (containing 0.01%                                                    of Px B)                                                                      0.2% Aqueous Emalgen 709                                                                        >90       --                                                solution (containing 0.01%                                                    of Px B)                                                                      0.4% Aqueous Amphitol 20N                                                                       >90       --                                                solution (containing 0.01%                                                    of Px B)                                                                      ______________________________________                                    

As is clear from the results shown in Table 6, the ET activity of S.typhosa 0901 LPS spiked to plasma is completely inhibited by thesimultaneous presence of each surfactant and Px B.

EXAMPLE 9

[Reagents]

The same AL solution, aqueous surfactant solutions and aqueous Px Bsolutions as described in Example 1 were used.

Aqueous β-glucan solutions

After 108 mg of curdlan [a linear (I+3)-β-D-glucan derivative, mfd byWako Pure Chemical Industries, Ltd.] was weighed, 10 ml of distilledwater for injection and then 800 μl of 1N NaOH were added to dissolvethe curdlan, whereby a 10 mg/ml solution was prepared. This solution wasdiluted 10 times with distilled water for injection to obtain a startingsolution, which was properly diluted with distilled water for injection.The thus obtained solutions were used as aqueous β-glucan solutions.

[Procedure]

By diluting 100 μl of 10 ng/ml aqueous β-glucan solution 10 times with900 μl of each of aqueous surfactant solutions having predeterminedconcentrations and containing 0.01 w/v % of Px B, there were preparedsolutions containing spiked β-glucan (the final concentration ofβ-glucan was 1 ng/ml, and the final concentrations of Px B and thesurfactant in the β-glucan spiked solutions were 0.009 w/v % and 0.9 to4.5 w/v %, respectively). The recovery of β-glucan from the variousβ-glucan spiked solutions was determined in the same manner as describedin Example 6.

[Results]

The results obtained are shown in FIG. 6. FIG. 6 is a graph obtained byplotting the recovery of β-glucan on the axis of ordinate correspondingto individual surfactant concentrations (in the β-glucan spikedsolutions containing 0.009 w/v % of Px B) on the axis of abscissa.

As is clear from the results shown in FIG. 6, the recovery of β-glucanwas kept constant at about 90% even when the surfactant concentrationwas increased. From this fact, it can be judged that in the abovesurfactant concentration range, the surfactant (polyoxyethylene glycolp-t-octylphenyl ether) neither inhibits nor enhance the reaction of ALsolution with β-glucan.

EXAMPLE 10

[Reagents]

There were used the same AL solution, aqueous surfactant solutions andaqueous Px B solutions as described in Example 1.

[Procedure]

By diluting 100 μl of ET solution having a predetermined concentration10 time with 900 μl of distilled water for injection, 0.2 w/v % aqueoussurfactant (polyoxyethylene glycol p-t-octylphenyl ether) solution, eachof 0.002 to 0.01 w/v % aqueous Px B solutions, or each of 0.2 w/v %aqueous surfactant (polyoxyethylene glycol p-t-octylphenyl ether)solutions containing 0.002 to 0.01 w/v % of Px B, there were preparedsolutions containing spiked ET (the final concentration of ET was 2ng/ml, and the final concentrations of Px B and the surfactant in the ETspiked solutions were 0.0018 to 0.009 w/v % and 0.18 w/v %,respectively). The ET concentration in the ET spiked solutions wasmeasured in the same manner as in Example 1.

[Results]

The results obtained are shown in FIG. 7. FIG. 7 is a graph obtained byplotting the gelation time (Tg) of the ET spiked solutions on the axisof ordinate corresponding to individual Px B concentrations in the ETspiked solutions on the axis of abscissa. In FIG. 7, -Δ- shows theresults obtained for the ET spiked solutions prepared by use of theaqueous Px B solutions various in their concentrations, and -□- showsthe results obtained for the ET spiked solutions prepared by use of theaqueous Px B solutions containing 0.2 w/v % of the surfactant.

As is clear from the results shown in FIG. 7, the ET activity of E. coli0.55: B5 LPS in the ET spiked solutions is completely inhibited by thesimultaneous presence of the surfactant and Px B.

EXAMPLE 11

[Reagents]

There were used the same AL solution, aqueous surfactant solutions andaqueous Px B solutions as described in Example 1, the same ET solutionsas described in Example 8, and the same β-glucan solutions as describedin Example 9.

[Procedure]

To 1.0 ml of normal human heparinized plasma was added 10 μl of 4 g/mlET solution and 10 μl or 100 ng/ml β-glucan solution, and 100 μl of theobtained plasma containing spiked ET and spiked β-glucan was diluted 10times with 900 μl of distilled water for injection, 0.2 w/v % aqueoussurfactant (polyoxyethylene glycol p-t-octylphenyl ether) solution, 0.01w/v % aqueous Px B solution, or 0.2 w/v % aqueous surfactant(polyoxyethylene glycol p-t-octylphenyl ether) solution containing 0.01w/v % of Px B. Each of the thus obtained diluted solutions washeat-treated at 80° C. for 5 minutes and then immediately cooled withice (the final concentrations of ET and β-glucan were 3.92 ng/ml and 196pg/ml). The β-glucan concentration in the diluted plasma thus obtainedwas measured in the same manner as in Example 6. The same procedure asdescribed above was carried out for a mixture prepared by adding 10 μlof distilled water for injection and 10 μl of 20 ng/ml β-glucan solutionto the same plasma as used in the above, and the β-glucan concentrationin the mixture was measured.

Further, on the basis of the measurement results, the recovery ofβ-glucan was calculated.

[Results]

The results obtained are shown in Table 7.

                  TABLE 7                                                         ______________________________________                                                      Recovery of β-glucan (%)                                                   ET spiked  Plasma without                                     Diluent         plasma     spiked ET                                          ______________________________________                                        Distilled water for                                                                           54200      107                                                injection                                                                     0.2% Aqueous surfactant                                                                       3540       98                                                 solution                                                                      0.01% Aqueous Px B                                                                            893        105                                                solution                                                                      0.01% Aqueous Px B                                                                            117        112                                                solution (containing 0.2%                                                     of the surfactant)                                                            ______________________________________                                    

As is clear from the results shown in Table 7, β-glucan can be measuredwithout the influence (the influence of causing positive errors inmeasured values) of ET present in a sample only when the surfactant andPx B are present during the measurement of β-glucan.

As is clear from the above, this invention provides a process forinhibiting ET activity in a sample effectively. The process of thisinvention is effective in that it permits easier and more certaininhibition of ET activity than do conventional processes, so that itmakes it possible to measure an ET analogous substance without theinfluence of ET. Therefore, this invention contributes greatly to theart.

What is claimed is:
 1. A process for measuring at least one endotoxinanalogous substance present in a sample and having a property ofreacting with a horseshoe crab hemocyte lysate to cause enzymeactivation reaction or gelation reaction, which comprisesi) pre-treatingthe sample having endotoxin and the endotoxin analogous substancetherein by selectively inhibiting the activity of endotoxin comprisingcontacting at least one surfactant and an endotoxin-inhibiting peptidewhich binds to the endotoxin to inhibit the reaction of the endotoxin,with the sample, then II) reacting the sample with horseshoe crabhemocyte lysate, and III) measuring the activity of an enzyme activatedby said enzyme activation reaction or measuring the degree of turbiditychange or gelation due to said gelation reaction caused by the reactionof step ii), by means of a measuring instrument or with the naked eye.2. A process according to claim 1, wherein the endotoxin analogoussubstance(s) is at least one member selected from the group consistingof (1-3)-β-D-Glucan and a derivative thereof.
 3. The process of claim 1wherein the endotoxin-inhibiting peptide is selected from the groupconsisting of polymyxins A, B, C, D, E, K, M and P, tachyplesin,polyphemusin and anti-LPS factor.
 4. A reagent for measuring at leastone endotoxin analogous substance in a sample which comprises anendotoxin-inhibiting peptide, at least one surfactant and AL solution.5. A reagent according to claim 4, wherein the endotoxin-inhibitingpeptide is polymyxin.
 6. A reagent according to claim 4, wherein thesurfactant is a nonionic surfactant or an amphoteric surfactant.
 7. Areagent according to claim 4, wherein the endotoxin analogoussubstance(s) is at least one member selected from the group consistingof (1-3)-β-D-Glucan and a derivative thereof.